Using molecular, biochemical and structural approaches, we have contributed to defining how specific human BER proteins recognize and process target lesions, as well as coordinate with other components of the pathway. This research has centered largely on apurinic/apyrimidinic endonuclease 1 (APE1), the major mammalian protein for repairing abasic sites in DNA, and x-ray cross-complementing 1 (XRCC1), a non-enzymatic scaffold protein that facilitates the efficient execution of SSB repair. Our most recent work has found that (i) in addition to abasic sites in conventional duplex DNA, APE1 has the ability to incise at AP sites in DNA conformations formed during DNA replication, transcription, and class switch recombination, and that APE1 can endonucleolytically destroy damaged RNA;(ii) in two- or multiple-lesion clusters typically associated with ionizing radiation exposure, the spatial arrangement of AP sites is key in determining APE1 cleavage efficiency;(iii) Mg2+, a divalent metal required for APE1 incision activity, likely binds a single site within the protein, in the absence of or when in complex with substrate DNA;(iv) the flap-endonuclease FEN1 plays a role in repairing mitochondrial oxidative DNA damage through a novel long-patch BER pathway;and (v) RECQL4, a human RecQ helicase mutated in approximately two-thirds of individuals with Rothmund-Thomson syndrome (RTS), regulates BER capacity both directly and indirectly. APE1 is the major mammalian enzyme responsible for the repair of abasic sites in DNA, and has functions in SSB repair and other cellular processes, including transcriptional regulation. We are in the process of establishing stable shRNA knockdown cell lines to dissect out the precise contribution of each proposed function of APE1 (i.e. its nuclease activity, redox regulatory role, etc.) in cell growth/viability and protection against DNA-damaging agents. The underlying hypothesis is that APE1, and BER more generally, plays a critical role in dictating cellular responsiveness to genotoxic insults and susceptibility to disease manifestation, particularly in the face of certain environmental exposure(s). Defining which of the many reported functions of APE1 are critical to normal cellular activity is a key step towards understanding the potential relationship of the protein to disease risk. XRCC1 is a non-enzymatic scaffold protein that appears to orchestrate a DNA repair response at sites of SSBs. Our efforts will continue to evaluate the contributions of XRCC1 to DNA damage responses, genomic stability and telomere maintenance in defined human cell lines using a chronic shRNA knockdown strategy. Our hypothesis is that defects in SSBR will be more deleterious to non-dividing, terminally-differentiated cells (e.g. neurons) than dividing cells, because dividing cells repair SSBs not only by SSBR, but also by replication-mediated HR. Such studies on XRCC1 will go a long way towards elucidating the molecular mechanisms underlying cancer, aging and neurodegenerative disease. DNA interstrand crosslinks (ICLs) can arise from reactions with endogenous chemicals, particularly the lipid peroxidation product malondialdehyde, as well as from exposure to a variety of clinical anti-cancer agents, such as bifunctional alkylators. Recent studies suggest a role for BER in the processing of these highly toxic DNA intermediates, which are absolute blocks to transcription and replication, and thus, lethal adducts. We are developing strategies to begin to explore the molecular involvement of specific BER factors in ICL removal. Defining the roles of BER proteins in ICL resolution may reveal new targets relevant to anti-cancer treatment paradigms.